Distal infrapyramidal and longitudinal mossy fibers at a midtemporal hippocampal level

BrrrinRrsecr~h &&fin, Vol. 10, pp. 137-146, 1983.Printed in the U.S.A.

Distal Infrapyramidal and Longitudinal Mossy Fibers at a Midtemporal Hippocampal Level JAMES

Department

of Anatomy,

College of ~e~~c~ne, Un~vers~iy of Iowa, Iowa City, IA 52242 Received

WEST, J. R. Disrol

infiupyrumidul

R. WEST

und longirudinul

28 May 1982

mossy.fihrrs

of (I mid~emporul

Icvt~l. BRAIN

hippocampal

RES BULL

10(1)137-146, 1983.-Timm’s and horseradish peroxidase histochemical methods were used to demonstrate a small, distal infrapyramidal mossy fiber projection at midtemporal hippocampal levels in the rat. The Timm’s sulfide silver method revealed sparse granules of dark, mossy fiber-like staining at an infrapyramidal location in an area roughly equivalent to hippocampal subfield CA3a (close to the CA1 border). The use of anterograde transport of horseradish peroxidase enabled the visualization of short mossy fiber axons that left the suprapyramidal bundle, penetrated the pyramidal ceil layer, and terminated in a distal inf~pyramida1 position. The HRP-labeled axons possessed periodic swellings that are characteristic of mossy fiber mo~hology. The HRP studies also provided evidence for lon~itudin~ly oriented mossy fibers at midtemporal hip~camp~ levels. The distal tip of the suprapyramidal band of mossy fibers turned tem~rally and could be detected up to 1140 micrometers ventral to the main suprapyramidal band. Rat

Hippocampus

Infrapyramidal

mossy fibers

THE mossy fiber axons of the granule cells represent the major excitatory pathway connecting the dentate gyrus with the hippocampus [4,5]. They project mainly via a suprapyramidal bundle to stratum lucidum, where they make connections en passrrnt on the proximal portions of large pyramidal cell apical dendrites [2, 3, 7, 8, 201. While small proximal intra- and infrapyramidal mossy fiber bundles exist adjacent the hilus, the classical mossy fiber terminal zone was thought to be restricted to stratum lucidum throughout the remainder of field CA3. The unusual mossy fiber terminals contain remarkably high levels of zinc 191, which can be stained with the Timm’s sulfide silver method for both light [ 10, 18,21,25,27, 29, 3 11 and electron microscopic studies [13]. At the ultrastructural level zinc was found only in the presynaptic terminals [13]. During the past few years several laboratories have used modifications of the Timm’s sulfide silver histochemical method to demonstrate evidence for a normal distal (extra hilar) infrapyramidal mossy fiber projection IlO, 25,281. This projection is restricted topographically to approximately the rostra1 (septal) one-third of the hippocampus. Recent studies from our laboratory 1271 and others [16181 have indicated that a variety of pre- and postnatal manipulations result in remarkably similar developmental alterations in mossy fiber topography; the distal infrapyramida1 terminal field extends well beyond its normally restricted position in the rostra1 one-third of the hippocampus into midtemporal hippocampal levels. The distinctive alterations in mossy fiber topography in each of those developmental studies were restricted to regio inferior. Lesion-induced sprouting in the hippocampus usually occurs as a consequence of an extension of other nondamaged afferent fibers in the

Copyright

o 1983 ANKHO

I~te~ational

Anterograde transport __-._

HRP

immediate vicinity of the partially deafferented neurons [12,30]. Such findings suggest that one factor which may

influence the similar aberrant mossy fiber changes /16-18,271 is the presence of a normally occurring, sparse PrOjeCtiOn of mossy fiber terminals in the vicinity of the deeper-lying

CA3a pyramidal cells. The purpose of this study was to investigate whether any distal infrapyramidal mossy fiber axons were present in normal rats at midtem~~l hippocampal levels where aberrant mossy fiber topog~phy has been reported. METHOD

A total of 18 adult Sprague-Dawley rats (King Animal Labs, Oregon, WI), eight processed with the Timm’s technique and ten with the anterograde horseradish peroxidase (HRP) method, were used for this study. A sensitive modification of the Timm’s sulfide silver stain for heavy metals was used [29]. The rats were anesthetized with an overdose of sodium pentobarbitol and perfused with 500 ml of a solution containing 5.85 g of sodium sulfide and 5.95 g of sodium phosphate monohydrate in 500 ml of distilled water, followed by a similar volume of 1.0% (w/v) parafo~aldehyde and 1.25% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). The fixative was followed by 100 ml of phosphate buffer to which 30% (w/v) sucrose had been added. Following perfusion the brains were removed and post-fixed for 12-24 hours at 4°C in the same sucrose-buffer. The brains were then sectioned horizontally (parallel to the dorsal surface of the hemispheres) at 30 FM with a freezing microtome, mounted on chrome-alum subbed slides, and stained on the slides by immersion in the developing solution for 60-75

Inc.-0341-9230/83/010137-1~03.00/0

138

WES’I

FIG. 1. Low power pbotomic~graph of a horizontal section through the midtemporal hippocampus stained with a mod Timm’s sulfide siker hist~bem~al method. Neavy Timm’s s~n~n~ correspon to the sup hatid @ilied arrows) can be seen in stratum lucidum but no intense m”s staining rapyra mossy occurs on the infrap idal ~st~~rn ovens) side of the ~yram~al 41 laver except nkar the hilos (open arrows). The tissue was co~t~~t~~ with m~byle~e blue. The foI~w~~& abb~~iat~ons are used in this and sMbseq~nt figures: G=granule cell layer; H=hilus; L~C~st~tum iuc~dum; ~~stratum oriens: PYR=st~bm‘pyramidale: a. b. and c denote the general positions of CA3 subtields. Scale bar-SO0 PM.

INFRAPYRAMIDAL

AND LONGITUDINAL

MOSSY FIBERS

FIG. 2. Photomicrographs of the portion of the hippocampus near the fimbria fCA3a). (A) Timm’s-stained section taken from the same midtemporal level as in Fig. 1. ~ndividuai mossy fiber-like granules are present within the pyramidal cell layer and on the stratum oriens (infrapyramidal) side, deep to most of the pyramidal cells. (B) Photomicrograph of HRP-labeled mossy fibers in a hippocampal section at the same midtemporal level as in A. The HRP-labeled mossy fiber terminal band in stratum lucidum is the classical suprapyramidal bundle. Note its topographical similarity to the dark staining observed in the Timm’s stained material in A. Counterstained with thionin. Scale bar=100 FM.

FIG. 3. Low power photomicrog~~h ofa NW-labeled mossy fiber prnjeetion at a m~tern~r~l hip~am~l level. Slight damage from the pipette tip can be seen in the media4 (intrapyr~id~l~ graphic ~~~~~~atinn of the mossy fiber t~~ina4 f& is biade of the dentate gyps. The F staining in Fig. I. The tissue was ~ounter~tained with similar to the suprapyramid~l Ti thiooin. Scale bar=SQO FM. FACING PAGE: FIG. 4. ~ark~e4d ~~torn~cro~~phs ii she y of the lon~~tud~ally coursing ASP-ladled mo.ssy fiber t~rmin~4 band, The fo4lowing m~c~$~phs are a series of p ions taken of sub~e4d CA3a (in the I& h4pp~a~pus~ from the sent an&al as 1YQ in Fig. 3. iA) From the same level as Fi y mossy fiber terminal 4a~4i~g was present ~ontin~sf~~~rn the hilus. (Fit From a ievel300 At this point only a very few labeled axons were observed in the hilus, CA3c or CA3b. By the next sect~oo no axons were iy to CA3a. (C) From a fevei 240 PM ventral to Thorsection in B. The entire remanning distal ~~i~~n of the ~~F-la~4~ rxmssy Id is visible. (D) From a level of 240 uM ventral to C 1480 PM ventral to 3). The labeling is sti41strong but the termina4 &&d is t this level the now reduced to only the distal one-half of subfield CA&. (E) From a level 240 &M ventral to D (720 MM to the section in pM beAow the terminal band continues to shrink but maintains its position near the tip of CA3a. W) From a 4~~1 240 J&I ventral to E section in B). The terminal field continues to decrease in size hut is still densely labeled at the tip. (G) From a level 480 ,&I ventral to F (I MO pM belaw the section in B). At t&s lint the 4~~~tudina4 projection is barely d~sce~ab~ even with d&k Geld i?i~~i~~ti#n” No sign of mussy fiber termins labeling was observed in more ventral sections. Scale bar =7,OO$%t.

INFRAPYRA~IDAL

AND LONGITUDINAL

MOSSY FIBERS

141

FIG. 5. Law power ph~tomi~r~~h of a midtemporal harizontal section through the htpp2cam pus showing the tip of the mossy fiber band labeled with antelope WRP. The section was ~~l~e~ 300 FM ventral to the point where the supra~yt~midal mossy fiber band was continuous huh the bilus. Note the mossy fiber ~o~ate~~s fwith h&eled s~ii~g~ cros~~~~ the ~yra~d~l cell layer (arrows). The tissue was co~nte~tai~ed with thionin. Scale bar-200 PM.

minutes in the dark at room tem~rature. The developing soIution consisted of 60 ml of 5 (w/v) gum arabic in double distilled water, 10 ml of 2 M citrate buffer (pH 4.0), 30 mlof 5.67% (w/v) hydr~uinone and 0.5 ml of 17% (w/v) Giver nitrate. The slides were then rinsed in running tap water for 10 minutes, dehyd~ted, cleared in toluene and coverslipped with Eukitt. Alternate sections were counterstains with thionin. The ant~ro~ade HRP rn~th~ 1261 w added by Small ionsh~~ening the survival time to 12-22 hours tophoretic injections (1.8 PA for 30 to 90 SK) of 20% (w/v) HRP (Sigma, type VI) containing 2% (w/v) dimethylsutfoxide (DMSO) with 1% (w!v) ~lyv~nylpy~iidone (PVP, added to Eimit the spread of the HRP at the injection site) in double distilled water were made in th entate gyrus. The injechip~ampal levels (detions were placed at midtempo fined here as the region just ve~r~l to the occipital bend of the hippocampus), Horizontal frozen sections (either 30 or 40 FM thick) were processed for HRP using tetramethyl ben&dine according to the method of Mesulam f22] except that only 0.4 ml of 1% hydrogen peroxide was used for the reaction 129). This modi~cat~~ helped to min~mjze ~~~facts so that mossy fiber axonaf trajectories could be followed more easily. The ~RP-ia~led tissue was counterstained with either safranin 0 or thionin [I].

Dense, dark brown staining was observed in the hilus and stratum lucidum that was characteristic of the welt docu-

fiber terminal &r&s in regio inferiar. The hilar tip oft cell layer (CA3c) exhibited i&r&- and fiber stain~g in addition to sypr~py~m~dat stacks (Pig. I). The distal infrapyramidal portion of regio inferior (roughly equivalent to bi~~mpai suh~%ld CA3a [ZO]), was devoid of heavy staining at leveb ventral to the ~ccjpit~ bend in all eight T~mm’s-staged brains. ~~~~ver, intermittent s~~n~~ in the form of small, i~e~~~r~~. shaped, dark. brown granules, was present in that re OXI(Fig. 2AL The j~dj~~~ua~ granules were variable in size, but many were s~rn~~~ in diameter to mossy fiber swellings stained with other tech-

the staling. However, with the Timm‘smethod it was not possible to detect whether mossy fiber axons ~presurn~~ls responsible for the srd From the sup?apyramidal bundle, or ma midd course f-rbm the hilus, to reach the st~~~rn oriens side of the ~~~~~dal celf layer.

tion to the mossy fiber tu~ap~y r~v~~~d with the ~~~rn~s method, The distal end of the sup~p~~rnid~l band tapered

INFRAPYRAMIDAI..

AND LONGITUDINAL

MOSSY FIBERS

143

FIG. 6. Photomicrographs of mossy fiber axons in subfield CA3a of the left hip~ampus labeled by the anterograde transport of horseradish peroxidase. Many mossy fiber axons with &polar and fusiform expansions are labeled in the suprapyramidal bundle in stratum lucidum. (A) Photomicrograph of HRP-labeled mossy fiber swellings in the distal position of CA3a. The short terminal portion of the mossy fiber axons (possibly collaterals) remain perpendicular to the pyramidal cell-layer. Note that one swelling was labeled that exhibited a ring (arrow) which may have circumscribed a pyramidal cell dendritic shaft. A second axon with a mossy fiber swelling (arrowhead) was also heavily labeled. The tissue was counterstained with thionin. Scale bar=75 ,uM, (B) At a more proximal position, a few mossy fiber axons have left the suprapyramidal band and penetrated the pyramidal cell layer to arrive at an infrapyramidal position (arrows). The characteristic swellings are similar in shape and size to those on the mossy fiber axons in the suprapyramidal bundle. The tissue was counterstained with thionin. Same magni~cation as A.

to a point which was less densely labeled than the rest of the labeled terminal field. The tip of this very lightly labeled area (occasionally a single labeled axon) sometimes reached what appeared to be the proximal border of regio superior. Analysis of serial sections revealed that the suprapyramidal mossy fiber band was oriented transverse to the long axis of the hippocampus. The transverse band was slightly wider in its septo-temporal width at the distal (CA3a) than proximal (CA3c) portion of regio inferior, giving it a slight temporal deviation. Furthermore, once it reached the distal portion of regio inferior, the projection turned sharply in the temporal direction and coursed for a considerable distance parallel to the longitudinal hippocampal axis. Therefore, at levels temporal to the injection site, all of the mossy fiber labeling was in the distal portion of regio inferior (near the regio superior border}. In the three most heavily labeled cases the distal tip

pursued a longitudinal course for over a millimeter more ventrally than the transversely oriented mossy fiber bundle (Fig. 4). The terminal area of the longitudinal band decreased with the distance from the transverse band, but remained confined to the most distal part of regio inferior. When small injections were combined with survival times in the range of lf&20 hours, much of the HRP was observed in the axons themselves, rather than in the terminal band [28,29]. This permitted the axonal trajectories of the mossy fibers to be examined. HRP-labeled suprapyramidal mossy fiber axons exhibited characteristic periodic swellings along their length (Figs. 6 and 7). In addition to labeling mossy fiber axons in the suprapyramidal bundle, the HRP injections succeeded in labeling discrete mossy fiber axons that left the suprapyramidal bundte and penetrated the pyr~idal cell layer (Figs. 6 and

FIG. 7. HRP-labeled mossy fibers in the right hippocampus following a large injection into the dentate gyrus ;rt a midtemporal hippocampal level. Individual mossy fiber axons. with characteristic swellings, can be seen brcaking away from the suprdpyramidal bundle in CA3a (on the left) penetrating the pyramidal cell layer I(, occupy it more distal infrapyramidal position as they course toward the regio superior border (to the right). 0ther short segments of mossy fiber axons can be seen. Some pyramidal cells are deep to the infraiintrapyramidal mossy fibers. The laraer iniection accounts for the lareer number of labeled mossy fiber axons compared IOFig. 5. Scale bar=SOuM. ”

Relatively few distal infrapyramidal mossy fiber axons were labeled in each section. However, a few axons in that region were labeled regardless of the location of the injection in the dentate gyrus. Two distinct forms of the terminal portion of mossy fiber axons were observed. The first type of axon branching from the suprapyramidal bundle in CA3a was usually the longer of the two forms. After penetrating the cell layer it coursed distally, in an infrapyramidal position parallel to the pyramidal cell layer, toward the regio superior border (Figs. 6B and 7). The second type of mossy fiber axon left the suprapyramidal band even farther from the hilus (i.e., closer to the regio superior border). It was typically short and maintained a position that was frequently perpendicular to the pyramidal cell layer (Figs. 5 and 6A). Both types of HRP-labeled axons possessed easily recognizable mossy fiber swellings. It could not be determined conclusively if these terminal axons were collaterals from axons that continued in stratum lucidum. Ventral to the main (transverse) suprapyramidal mossy fiber band short mossy fibers were occasionally observed branching from the longitudinal bundle (Fig. 6). 7).

DISCUSSlOlr;

The Timm’s sulfide silver method demonstrates the presence of heavy and transitional (group IIb) metals by staining the tissue. The staining intensity of the tissue indicates different concentrations of various stainable metals [ 14.3 11. The granules in the distal infrapyramidal region were similar

in color, density, and size to other mossy fiber terminal staining suggesting the presence of mossy fiber axons from the granule cells in the dentate gyrus. However, the granules were not contiguous with the suprapyramidal staining, so that conclusion was equivocable without confirmation using a technique that labeled the mossy fiber axon trajectories. In combination however. the Timm’s and HRP experiments demonstrated that a sparse mossy fiber projection does reach distal infrapyramidal locations at midtemporal levels of the rat hippocampus. In addition, the anterograde HRP labeling indicated that those few granule cell axons that pursue a distal infrapyramidal course at midtemporal levels possess typical mossy fiber morphology. There were fewer HRP-labeled mossy fiber swellings than Timm’s-stained granules, but both techniques indicated the presence of more infrapyramidal terminals near the distal than proximal portion of CA3a. The anterograde HRP method also demonstrated the presence of infrapyramidal mossy fiber swellings at levels of CA3a temporal to the injection site. Substantially fewer infrapyramidal mossy fibers were labeled with the HRP than the Timm’s stain, no doubt reflecting variables of the active transport technique, such as size and location of the injection and survival time. Not surprisingly, large injections that were not confined to a specific limb of the dentate gyrus labeled more distal intra- and infrapyrmidal mossy fiber axons than the smaller injections (compare Figs. 6 and 7). The additional mossy fiber innervation from the longitudinal mossy fibers (Fig. 5). diverging from more septal levels, also must contribute to the appear-

INFRAPYRAMIDAL

AND LONGITUDINAL

MOSSY FIBERS

145

FIG. 8. Drawing of the midtemporal hippocampal level summarizing the organization of transverse and longitudinal mossy fiber projections labeled after a small HRP injection. Once the mossy fiber axons reach CA3a they abruptly turn ventrally and run parallel to the septo-temporal axis as longitudinal mossy fibers. Collaterals which account for the sparse distal infrapyramidal innervation branch from the suprapyramidal mossy fiber bundle. The most distal of the infrapyramidal mossy fibers at this septotemporal level do not appear to course longitudinally as a separate infrapyramidal bundle, but reach more ventral levels only as short collateral branches from the distal tip of the primary (suprapyramidal) bundle

ante of more Timm’s granules in CA3a than in subfield CA3b (Fig. 8). Therefore, of the two techniques, the Timm’s

method is likely to be a better indicator of the actual number of mossy fiber boutons in that region. The confirmation of a normal, albeit sparse, distal infrapyrmidal mossy tiber projection may have important implications concerning possible mechanisms involved in the strikingly similar organization of aberrant infrapyramidal terminal field observed following various manipulations during hippocampal development. Dense distal infrapyramidal terminal terminal fields occur in regio inferior following exposure to ethanol in utero [27], after early postnatal treatment with L-thyroxine [16,17], or partial hippocampal lesions [18]. The sparse normal mossy fiber population of CA3a is in a position to proliferate following these manipulations. However, there are also Timm’s stained granules in subfield CA3b. It is puzzling, therefore, that although aberrant mossy fibers sometimes occur in CA3b [17] (and in preparation), CA3b appears to be less influenced than CA3a by developmental manipulations. The functional significance of the small population of distal infrapyramidal mossy fibers is obscure. However, a recent study of Lipp and his associates indicated a positive correlation between the size of the infrapyramidal mossy fiber terminal field and poor two-way avoidance performance [24]. Interestingly, in utero ethanol exposure causes extra distal infrapyramidal mossy fibers [27] and similar behavioral deficits ]19,23].

The present study also raises some interesting points concerning the field of influence of the mossy fiber axons. The HRP material indicated that the distal tip of the mossy fiber terminal band sometimes reached the proximal end of the small pyramidal cells. However, the distal end of regio inferior (the so-called CA2 field) contains some small pyramidal cells as it approaches regio superior 1201. Therefore, it is difficult to determine in the Nissl stained tissue if there is a distinct CA2 field or if the mossy fibers actually reach to the edge of regio superior. In agreement with others [6, 11, 21, 25,28,29], the main course of the suprapyramidal mossy fiber projection was observed as a narrow band roughly pkrpendicular to the longitudinal (septo-temporal) hippocampal axis. However, the HRP material indicated that even at midtemporal levels the distal tip of the mossy fibers turns sharply in the temporal direction and courses for a considerable distance as a narrow longitudinal band. The rostral, and to a lesser extent, the temporal one-thirds of the hippocampus are known to have temporally oriented mossy fibers [10,25]. The mossy fibers in the middle one-third of the hippocampus were thought to project transverse to the long axis or to have only a slight temporal deviation [6,10]. The more extensive longitudinal projection of midtemporal mossy fibers reported here (which is in agreement with recent observations derived from experiments using retrograde labeling techniques [l l]), may reflect greater sensitivity of the anterograde HRP method compared with silver degeneration techniques [6,10]. The markedly temporal orientation of the

Wr,SI

I46

distal tip of the mossy fiber band suggests considerable divergence of the mossy fiber system, but only in a restricted portion of distal regio inferior. It is nateworthy that ather lo~itudin~ fibers have been observed in this small area of the hippocampus [2%2SJ. Mossy fibers normally terminate on the proximal apical dendritic shafts of pyramidal cells 12, 3, 7, 201. In case\ where distal infrapyramidal mossy fibers normally occur, they may make contact with apical dendrites of the deeper-lying pyrmidal cells or perhaps with basal dendrites of more superficial neurons as well. Mossy fiber swellings usually develop before the postsynaptic specialization and terminate on later forming specialized dendritic “thorny excrescences” [2, 3, 7, 201, even when they do make contact with basal dendrites as in the hilus 121. It is not known whether the

1. Adams. J. C. Stabilizing and rapid thionin staining of TMRbased HRP reaction product. .Vturrm.i Lerr 17: 7-9. 1980. 2. Amaral, D. A golgi study of cell types in the hilar region of the hippocampus of the rat. .I Co,np /\‘crtro/ 182: 85 I-914. 1978. 3. Amaral, D. B. and J. A. Dent. Development of the mossy tibers of the dentate gyrus: 1. A light and electron microscopic study of the mossy fibers and their expansions. .I romp Ncurr~f 195: 51-86. 1981. 4. Andersen. P. Organization of hippocampal neurons and their connections. In: 7”/1r Hippoccrmpus, Vol. I. edited by R. I_.. Isaccson and K. H. P&ram. New York: Plenum. 1975. pp. 15-S-185. 5. Andersen. P., T. V. P. Bliss and K. K. Skrede. LamelIar organization of hippocampal excitatory pathways. E.rp Hnrirr Krs 13: 222-238, 1971. 6. Blackstad. T. W.. K. Brink. J. Hem and 3. Jeune. Distribution nf hippocampal mossy fibers in the rat. An experimental study with silver impregnation methods. .I c’ortrp ilieurol 138: 433-450. 1970. 7. Blackstad, T. W. and A. Kjaerheim, Special axo-dendritic synapses in the hippocampal cortex: Electron and light microscopic studies on the layer of mossy fibers. J Cmtp Nruroi 117: 133-159, 1961. 8. Cajal, S. Ram& y. Estructura del asta de Ammon. Ancri .Sl?c E.sp Wisr Nat Mtrdr 22: 53-l 14. 1893. 9. Danscher, G.. E. J. Fjerdingstad, E. Fjerdingstad and K. Fredens. Heavy metal content in subdivisions of the rat hippocampus (zinc, lead and copper). Bruin RPJ 112: 442-446, 1976. IO. Gaarskjaer, F. B. ~~~zation of the mossy fiber system of the rat studied in extended hip~amp~. II. ~x~~mental analysis of fiber distri~tion with silver impregnation methods. J Gmp Nvurol 178: 73-88, t978. 1I. Gaarskjaer, F. B. The hippocampal mossy fiber system of the rat studied with retrograde tracing techniques. Correlation between topographical organization and neurogenetic gradients. .! Cwnp Nertrd 203: 717-735. 1981. 12. Goldowitz. D., W. F. White. 0. Steward, 6. Lynch and C. W. Cotman. Anatomical evidence for a projection from the entorhinal cortex to the contralateral dentate gyrus of the rat. E$ h’eloz>l 47: 433-441. 1975. 13. Haug, F.-M. S. Electron microscopical localization of the zinc in hippocampal mossy fiber synapses by a modified sulphide silver procedure, Hisfochemir 8: 355-368, 1967. 14. Haua. F.-M. S. Light microscopial mapping of the hip~mpal regi&, the pyrifo~ cortex and the co~comediaI arny~~lo~d nuctei of the rat with Timm’s sulphide silver method. I. Area dent&a, hippocampus an subiculum. Z Antrt f+tlf~l+i, &vch 145: l-27. 1974. IS. Mine, R. J. and G. D. Das. Neuroembryogenesis in the hippocampal formation of the rat: An autogradiographic study. A Anor Enrbtkk-Gesch 144: 173-186. 1974.

distal infrapyramidal mossy fibers synapse only such postsynaptic specializations. This information is terest, especialty if the mossy fiber axons that form the rant terminal fields ~16-18, 271 possess classical mossy rno~~ol~~ and terminate on thorny excrescence\. few

with of inaberfiber

f thank Rwight Pierce and Cheryl Hodges-Savola for therr expert technical assistance, Paul Reimann, Bill Coons and Helen Fankhauser for their photoR~phic help. and 1,cslee M&r, Joyce Kline and Julie Anolik for typing the manuscript. The helpful conversations with Dr. Gary W. Van Hoescn :IIC also gratefully acknowledged. This work waz supparted by grams 4A 03884 and AA 05523 from the NIAAA.

16. Lauder, J. and E. Mugnaini. Earl) hy~rthyroidism dlterh the distribution of mossy fibers in the rat hippocampus. ,Vn/~rr 268: 335-337, 1977. 17. Lauder. 1. and E. Mugnaini. Infrapkrdmidal mossy fibers in the hippocampus of the hyperthyroid rat. no1 rli’crrrr8yc.i3: 248-265, 1980. 18. I-au&erg, S. and J. Zimmer. I,e~i~}n-induced rerouting of hippocampal mossy fibers in developing but not adult rats. .I C‘t~nr, !Y:(wo/ 190: 627-650, 1980. 19. Lochry. E. A. and E. P. Riley. Retention of passive avoidance and T-maze escape in rats exposed to alcohol prenatally. C’cwrohdwr~ ‘I;kriw/ 2: 107-l IS. 1980. 20. Lorente de N6, R. Studies on the structure of the cerebral cortex. If. Cc~nt~nuation of the study of the ammonic system. J P”$vrhoi Neurrtf 46: I IF- t77. 1934. ?I. McLardy, T. Some cell and fibre peculiarities of uncal hippocampus. Yrq Brairl Rcs 3: 71-88, 1963. 21. Mesulnm, M.-M. Tetramethylbenzidine for horseradish peroxidase histochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferentc and efferents. J Hirfrwham Cylr~chcm 26: 106-I I?, 1978. 23. Riley. E. P.. E. A. Lochry and N. R. Shapiro. lack of response inhibition in rats prenatally exposed to ethanol. p1.v~~~Jp~f~r~~~cohq~ (Berlin) 62: 47-32, 1979. 24. Schwegler, H.. H. P. Lipp. H. Van der Lao?, and W. Buselmaier. Individual hippocampal mo\sy fiber distribution in mice correlates with two-way avoidance performance. Sric,!rcc 214: 817-819. 1981. 25. Swanson. L. W., J. M. Wysu and W. M. Cowan. An authe or~a~tion of the tor~io~raphic study of int~hip~~~l association pathways in the rat. .I (“i?mp Nrurol 181: 681-716, 1978. 26. West, J. R. and A. C. Black. JI. Lnhancing the antcrograde movement of HRP to label sparse neuronal projectiom. !Vcrcr/l.sc.ii&r 12: 35-40, 1979. 27. West, J. R., C. A. Hodges and A. C. Black, JF. Prertatal ethartol exposure alters the o~i~tion of ~p~ampal mossy fiber-. St Gvtc~c~ 2 I 1: 957-959 1 t981 28. West, J. R.. C. A. Hodges and ~1. C. Black, Jr. Distal infrapyramidal granule cell axons possess typical mossy fiber morphology. &in Re.5 Bull 6: 119-124, 1981. 29. West. J. R., G. W. Van Hoesen and K. C. Kosel. A demonstra tion of hippocampal mossy fiber axon morphology using the ~nte~~~~e transport of horseradish peroxidase. Erp Nrrrin Hes, in press. 30. Zimmer. J. Proximity as a factor rn the re~ul~ti(~n of aberrant axonal growth in postnatally deafferented fascia dentata. fhin Rev 72: 137-142. 1974. 31. Zimmer. J. and F.-M. S. Haug. Laminar differentiation of the hippocampus, Fascia dentata and subiculum in developing rats. observed with the Timm sulphide silver method. J C#knzp,Veltntl 179: S81-618. 1978.